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| United States Patent |
5,098,823
|
|
Bodmer
, et al.
|
March 24, 1992
|
Chromosome-specific nucleic acid probe for familial Polyposis coli
Abstract
A nucleic acid fragment capable of selectively hybridizing with the human
chromosome 5 at the chromosomal region 5q20-q23 is disclosed. Also
disclosed are probes which include the fragment bearing a detectable level
as well as processes for presymptomatic screening for FAP and processes
for the pathological classification of colonic tumors and precancerous
polyps.
| Inventors:
|
Bodmer; Walter F. (London, GB);
Murday; Victoria A. (London, GB);
Bailey; Carolyn J. (London, GB);
Williamson; Robert (London, GB)
|
| Assignee:
|
Imperial Cancer Research Technology Ltd. (London, GB)
|
| Appl. No.:
|
460896 |
| Filed:
|
April 12, 1990 |
| PCT Filed:
|
August 11, 1988
|
| PCT NO:
|
PCT/GB88/00665
|
| 371 Date:
|
April 12, 1990
|
| 102(e) Date:
|
April 12, 1990
|
| PCT PUB.NO.:
|
WO89/01481 |
| PCT PUB. Date:
|
February 23, 1989 |
Foreign Application Priority Data
| Current U.S. Class: |
435/6; 435/252.3; 435/320.1; 436/501; 536/24.31 |
| Intern'l Class: |
C12Q 001/68; C12N 001/20; G01N 033/566; C07H 015/12 |
| Field of Search: |
435/6,253.3,320
436/501
536/26,27,28
935/77,78
|
References Cited
Other References
Nature, vol. 328, 13 Aug. 1987, W. F. Bodmer et al., pp. 614-616.
Nature, vol. 328, 13 Aug. 1987, E. Solomon et al., pp. 616-619.
Science, vol. 238, 4 Dec. 1987, M. Leppert et al., pp. 1411-1413.
Biological Abstracts/RRM, vol. 35, No. 64125, K. P. Meera et al.
(Switzerland) 1987, vol. 46, No. 1-4, p. 661.
Biological Abstracts /RRM, vol. 35, No. 64065, M. Leppert et al.
(Switzerland) 1987, vol. 46, No. 1-4, p. 647.
Genetic Analysis of Familial Polyposis Coli, Analysis of Gene Expression of
Oncogenes in Colon Tumors of FPC by Kenji Sugio, Midical Inst. of
Bioregulation, Kyushu University, Fukuoka 812, Japan, pp. 185-197.
The Gene for Familial Polyposis Coli Maps to the Long Arm of Chromosome 5,
by M. Leppert et al., Science, vol. 238, No. 4832, pp. 1411-1413.
|
Primary Examiner: Wax; Robert A.
Assistant Examiner: Zitomer; Stephanie W.
Attorney, Agent or Firm: Morrison & Foerster
Claims
We claim:
1. An isolated nucleic acid fragment capable of selectively hydridising
with the human chromosomes 5 at the chromosomal region 5q20-q23 other than
the probe C11p11.
2. An isolated double-stranded DNA fragment capable of selectively
hybridising under high stringency conditions with the human chromosome 5
at the chromosomal region 5q20-q23 other than probe C11p11.
3. A fragment according to claim 1 capable of selectively hybridising at
the chromosomal region 5q21-q22 locus.
4. A fragment according to claim 3 and capable of recognising alleles of
3.9 and 4.4 kb in a TaqI digest of human chromosomes 5.
5. A fragment according to claim 4, 3.6 kb in length.
6. A nucleic acid probe comprising a fragment according to claim 1 and
having a detectable label bound thereto.
7. A probe according to claim 6 wherein the label is a radiolabel, a
fluorescent label or an enzyme label.
8. A process for producing the probe C11p11 or a fragment according to
claim 1 comprising digesting human chromosomal DNA to form a library,
cloning the library and screening and selecting from the cloned library
fragments capable of selectively hybridising at the 5q20-q23 region of
human chromosome 5.
9. A process for presymptomatic screening of an individual for FAP
comprising contacting a sample of DNA from the individual with the C11p11
or a probe according to claim 6 under hybridising conditions.
10. A process for pathological classification of colonic tumors and
precancerous polyps comprising contacting DNA from support tissue sample
with the probe C11p11 or a probe according to claim 6 under hybridising
conditions.
11. A fragment according to claim 2 capable of selectively hybridising at
the chromosomal region 5q21-q22 locus.
12. A nucleic acid probe comprising a fragment according to claim 2 and
having a detectable label bound thereto.
13. A nucleic acid probe comprising a fragment according to claim 3 and
having a detectable label bound thereto.
14. A nucleic acid probe comprising a fragment according to claim 4 and
having a detectable label bound thereto.
15. A nucleic acid probe comprising a fragment according to claim 5 and
having a detectable label bound thereto.
16. A process for producing the probe C11p11 or a fragment according to
claim 2 comprising digesting human chromosomal DNA to form a library,
cloning the library and screening and selecting from the cloned library
fragments capable of selectively hybridising at the 5q20-q23 region of the
human chromosome 5.
17. A process for producing the probe C11p11 or a fragment according to
claim 3 comprising digesting human chromosomal DNA to form a library,
cloning the library and screening and selecting from the cloned library
fragments capable of selectively hybridising at the 5q20-q23 region of the
human chromosome 5.
18. A process for producing the probe C11p11 or a fragment according to
claim 4 comprising digesting human chromosomal DNA to form a library,
cloning the library and screening and selecting from the cloned library
fragments capable of selectively hybridising at the 5q20-q23 region of the
human chromosome 5.
19. A process for producing the probe C11p11 or a fragment according to
claim 5 comprising digesting human chromosomal DNA to form a library,
cloning the library and screening and selecting from the cloned library
fragments capable of selectively hybridising at the 5q20-q23 region of the
human chromosome 5.
20. A process for presymptomatic screening of an individual for FAP
comprising contacting a sample of DNA from the individual with the probe
C11p11 or a probe according to claim 7 under hybridising conditions.
21. A process for pathological classification of colonic tumors and
precancerous polyps comprising contacting DNA from suspect tissue sample
with the probe C11p11 or a probe according to claim 7 under hybridising
conditions.
Description
TECHNICAL FIELD
The present invention relates to nucleic acid fragments and their use in
the diagnosis and prognosis of adenocarcinoma especially Familial
Adenomatous Polyposis.
BACKGROUND OF THE INVENTION
Colorectal cancer is the second most common cancer in the United Kingdom
and other developed countries in the West. Although usually not familial
there is a rare dominantly inherited susceptibility to colon cancer,
Familial Adenomatous Polyposis (FAP or Familial Polyposis Coli). During
adolescence affected individuals develop from a few hundred to over a
thousand adenomatous polyps in their large bowel. These have a
sufficiently high probability of giving rise to adenocarcinomas that
prophylactic removal of the colon is recommended in diagnosed FAP
individuals. Polyps may also occur elsewhere in the gastrointestinal tract
and the condition is often associated with other extracolonic lesions,
such as epidermoid cysts, jaw osteomata and fibrous desmoid tumours.
.sup.1,2,3,4
Adenomata have been suggested to be precancerous states for the majority of
colorectal tumours .sup.5,6. Knudson.sup.7 has suggested that in both
familial and non-familial cancers, dominant genes give rise to cancer
susceptibility. He further proposed that these mutations act recessively
at the cellular level, and that both copies of the gene must be lost for
the cancer to develop. In sporadic cases both events occur somatically
while in dominant familial cases susceptibility is inherited through a
germ line mutation and the cancer develops following a somatic change in
the homologous allele. This model was first substantiated by the elegant
molecular work on retinoblastoma .sup.2a. Following the linkage of the
disorder to chromosome 13.sup.3a, Cavenee et al.sup.2a, using polymorphic
DNA markers for this chromosome, showed loss of heterozygosity in tumour
material compared to normal tissue from the same patient, in both familial
and non-familial forms. Similar results have now been obtained, following
cytogenetic evidence which suggested the localisation of the disease on a
specific chromosome, for Wilm's tumours .sup.4a-7a, acoustic
neuromas.sup.8a as well as several other tumours .sup.9a,10a.
DISCLOSURE OF THE INVENTION
It has now been discovered that a DNA probe designated C11p11 localises to
the chromosomal region 5q21-q22 and that this marker is closely linked to
the disease gene for FAP. Moreover 20 to 25% of sporadic cases of
adenocarcinoma of the colon involve changes in the human chromosome 5.
Accordingly the present invention provides a nucleic acid fragment capable
of selectively hydridizing with the human chromosome 5 at the chromosomal
region 5q20-q23.
In another embodiment, the present invention relates to a double stranded
DNA fragment capable of selectively hybridizing under high stringency
conditions with the human chromosome 5 at the chromosomal region 5q20-q23.
In other embodiments, the invention comprises processes for producing the
subject fragments, nucleic acid probes comprising the fragments carrying a
detectable label, processes for presymptomatic screening for FAP using
these probes and processes for pathological classification of colonic
tumors and precancerous polyps using the subject probes.
BRIEF DESCRIPTION OF THE FIGURES
Figure Legends
FIG. 1--Family 79 demonstrating segregation of FAP with a 3.9 Kb fragment
using the probe C11p11.
Methods
Blood samples were collected from family members and lymphocytes separated
and frozen for future transformation with EBV.sup.9. High molecular weight
DNA was extracted from both the residue of the separation and from the
transformed lymphocytes. The cells were lysed in a solution containing
0.33M sucrose, 5 mM MgCl, 10 mM Tris-HCl pH 7.5, 1% Triton X-100. Nuclei
were pelleted at 8000 g at 4.degree. C. for 10 mins. and resuspended in 75
mM NaCl, 25 mM EDTA, 100 .mu.g/ml Proteinase K and 0.5% SDS. The mixture
was incubated overnight at 37.degree. C. The aqueous phase was extracted 3
times in 2 mM Tris:2 mM EDTA buffered phenol and once in isoamyl
alcohol/chloroform (1:24). The DNA was reprecipitated with 1/10 volume 3M
sodium acetate pH 5.2 and two volumes absolute alcohol and then
resuspended in TE. 10 .mu.g aliquots of genomic DNA were digested
overnight with 50 units of retriction enzyme, under the recommended
conditions (Taq1 digests for 4-6 hours). The samples were run in 0.8%
(w/v) agarose gels for 36-38 hours, at 1-1.5 V/cm. The gels were
depurinated in 0.25M HCl for 10 mins, denatured in NaOH for 45 mins and
then neutralized for 60 mins. The DNA was then transferred on to Hybond-N
nylon filters (Amersham) by standard procedure.sup.16.
Probes were labelled with [.alpha..sup.32 P]dCTP by the oligolabelling
method.sup.17 for 4-6 hours at 37.degree. C. The filters were
prehybridised in 6.times. SSC, 0.1% SDS, 5.times.Denhardts solution
containing 25 .mu.g/ml of denatured salmon sperm DNA for 1-2 hours at
65.degree. C. Hybridisation was carried out overnight at 65.degree. C. in
the same solution but supplemented with 10 .mu.Ci/ml of labelled probe and
10% dextran sulphate.
L1.7 (D5S3.sup.10) is a 1.14 kb EcoRI fragment cloned into pBR322 and
detects a BglII polymorphism with alleles A1 at 7.9 kb and A2 at 10.6 kb.
L1.7 also detects a PsTI polymorphism with alleles B1 at 2.8 kb and B2 at
2.3 kb.
L1.4 (D5S4.sup.10) is a 0.72 kb EcoRI fragment cloned into pBR322 and
detects an EcoRI polymorphism producing alleles A1 at 0.7 kb and A2 a 0.6
kb.
L1.4 also detects an RsaI polymorphism with alleles C1 at 2.4 kb and C2 at
2.2 kb.
C11p11 is a 3.6 EcoRI fragment cloned into pUC8 and is polymorphic with
TaqI producing alleles A1 at 3.9 kb and A2 at 4.4 kb.
.lambda.MS8.sup.11 produces multiple alleles with HinfI.
HEXB.sup.10 is a 1.5 kb XbaI fragment cloned into pSP64. A 1.1 kb
BamHI/PstI fragment detects a PstI polymorphism with alleles A1 at 1.6 kb
and A2 at 1.0 kb.
FIG. 2a
Representative chromosome spread showing in situ hybridisation of probe
C11p11.
In situ hybridisation was performed on chromosomes prepared from
PHA-stimulated normal blood lymphocytes essentially as described by Harper
& Saunders.sup.20. The C11p11 probe was labelled using oligoprimers and
.sup.3 H-deoxyribonucleotides to a specific activity of 1.times.10.sup.7
cpm/.mu.g/ml. Slides were washed in 2.times.SSC at 39.degree. C.,
dehydrated and dipped in Ilford K-5 emulsion. They were developed after 10
days.
FIG. 2b--Fine chromosome mapping of probe C11p11 and putative regional
assignment of probes L1.4 and .lambda.MS8.
Fine chromosome mapping was carried out with C11p11 probe, 221 silver
grains were scored on chromosomes from 150 metaphases, with 22 grains
(9.9%) present over bands 5q15-23 and the majority located over bands
5q21-22. Preliminary data for in situ hybridisation of the L1.4 probe
localises it to band 5q31.
Localisation of .lambda.MS8 was done by in situ hybridisation by N. J.
Royle and A. J. Jeffreys (unpublished data).
The symbols .DELTA. regions represent the interstitial deletions reported
by Herrera et al.sup.8
Figure Legends
FIG. 3--Chromosome 5 allele loss in primary colorectal tumours and cell
lines
FIG. 3a--Primary colorectal tumours. DNA from matched normal/tumour pairs
hybridised with .lambda.MS8 and L1.4
Methods
DNA was extracted from tumour biopsies (T) and normal adjacent mucosa (N)
by standard methods.sup.29. For analysis with .lambda.MS8, DNA was
digested with HinFI (New England Biolabs) and fractionated by agarose gel
electrophoresis on 0.8% gels, for 60 hours at 20 mA; for L1.4 DNA was
digested with EcoRI (Boehringer Corp. Ltd.), fractionated on 1.2% agarose
gels, for 18 hours at 63 mA. In both cases DNA was transferred to Hybond-N
Nylon membranes (Amersham) by standard procedures.sup.30. Probes were
oligo-labelled.sup.31 with .sup.32 P-CTP. Hybridisation and washing were
by standard methods.sup.29. Patient numbers refer to Table 1.
FIG. 3b
Colorectal cell lines. DNA hybridised with .lambda.MS8. Methods are as in
1a. Cell lines are:
1) Coll2/23.sup.32, 2) Coll 5/3.sup.32, 3) HT29.sup.33, 4) LoVo.sup.34, 5)
LS174T.sup.35, 6) SV48.sup.36, 7) SW620.sup.36, 8) SW837.sup.36, 9)
SW1222.sup.36, 10) PCJW.sup.19, 11) PC/11.sup.19
FIG. 4--Densitometric Scanning of Autoradiographs with Normal and Tumour
DNA hybridised with .lambda.MS8
Autoradiographs were scanned with a Joyce Loebl Chromocan 3. Areas under
the peaks are plotted as the ratio of the two tumour alleles, Tp.sub.1
/T.sub.2, divided by the ratio of the two normal alleles Np.sub.1
/N.sub.2. Those 7 cases which could be seen as clear allele loss are
represented as .cndot. (left to right: patients 9, 18, 19, 37, 17, 38, 35)
and those with less obvious loss as o (patients 21 and 11).
DETAILED DESCRIPTION
The fragments of the invention may hybridise with the 5q20-q23 region under
low or high stringency conditions and will be selective in that the
fragments do not hydridise with other human chromosomal DNA under those
conditions.
The nucleic acid fragments may be RNA or DNA but the latter is preferred.
More preferably the fragment is double stranded DNA.
It is preferred that the present nucleic acid fragment will hydridize at
the 5q21-q22 locus.
Nucleic acid fragments according to the invention may be obtained for
instance by complete EcoRI digestion of human chromosome 5 (preferably
after sorting the chromosomes, for instance by flow sorting), cloning in a
suitable vector and amplification in a suitable host. Nucleic acid
fragments according to the invention are then obtained by EcoRI digestion
of the amplified vector and screening for hydridization to the 5q20-q23
region. This process has been used to prepare a 3.6 kb fragment which
hybridises at the 5q21-q22 region and which recognises alleles of 3.9 and
4.4 kb in a TaqI digest of human chromosomes 5. The fragment forms a
particular aspect of the present invention and has been designated C11p11.
Other nucleic acid fragments according to the invention include those which
are capable of hydridizing to a C11p11 fragment although they may be
longer or shorter than C11p11 provided that they still conform to the
requirement for selective hybridisation of 5q20-q23. Such fragments may be
produced by alternative digestions of human chromosome 5, by manipulation
of C11p11 or a portion thereof or by de novo nucleic acid synthesis based
on information obtained from sequencing for instance C11p11.
Further fragments according to the present invention are also capable of
hydridising in the 5q20-q23 region but do not hybridise with the C11p11
fragment. These may be obtained for instance by alternative digestions of
human chromosome 5 affording relatively long DNA fragments which will
hybridise with C11p11 but which also include flanking sequences and,
optionally after screening for C11p11 hybridisation and/or hybridisation
at 5q20-q23, are further cut to obtain a flanking sequence which does not
overlap with C11p11. This process may be repeated to obtain yet further
fragments which still hybridise in the 5q20-q23 region but which represent
flanking sequences more remote from the locus recognised by C11p11.
The invention also comprises a process for producing a fragment as
hereinbefore defined comprising digesting human chromosomal DNA, cloning
the library of fragments so obtained and screening and selecting fragments
which hybridise selectively at the 5q20-q23 region of the chromosome.
For use as probes it is necessary for the fragments of the invention to be
detectable after hydridisation with human DNA and this may be achieved by
any known labelling technique. Typically the probe will be labelled by
incorporation of radioisotopes (for instance by nick translation) which
may be detected by autoradiography. Other labelling techniques include
provision of directly detectable labels such as fluorescent labels and
indirectly detectable labels such as enzyme labels. A skilled person will
be aware of the techniques required for the introduction of these and
other known labels and for the detection of such labels.
The invention therefore also provides a probe comprising a nucleic acid
fragment as defined above having a detectable label attached thereto.
As discussed in more detail in the Examples, predisposition to FAP in
afflicted families can be detected by heterozygosity in the 5q20-q23
region especially the 5q21-q23 region and the probes of the invention may
be used to trace disease-related genes within families and to identify
at-risk individuals.
Accordingly the invention provides a process for presymptomatic screening
of an individual in an FAP family comprising contacting DNA from the
individual with a probe of the invention under suitable hybridising
conditions.
The process may be conducted as a pre- or post-natal screen to identify
at-risk foetuses or individuals within FAP families and preferably further
comprises screening relatives with and without FAP to identify and track a
disease-related allele.
The invention also provides a process for pathological classification of
colonic tumours and precancerous polyps comprising contacting DNA form a
suspect tissue sample with a probe of the invention under suitable
hybridising conditions. Identification of potential or actual tumours
involving a 5q20-q23 abnormality may assist in prognosis and selection of
appropriate treatment.
These processes may be conducted by standard methods such as by Southern
blotting .sup.30 using DNA from lymphocytes normal and/or tumour tissue
obtained in blood samples or by biopsy. A general description of such
restriction fragment length polymorphism (RFLP) analysis can be found in
Scientific American, August 1987, pp21-22.
Further details of the invention will be apparent from the following
Examples which are not intended to limit the scope of the invention in any
way.
EXAMPLE 1
A case report of an interstitial deletion of chromosome 5 in a mentally
retarded individual with multiple developmental abnormalities and
FAP.sup.8 promoted us to search for linkage of FAP to restriction fragment
length polymorphisms (RFLPs) revealed by DNA clones assigned to chromosome
5. Our data show that the FAP gene is on chromosome 5, most probably near
bands 5q21-22.
Most of the families for the study were provided by the polyposis register
at St. Marks Hospital, London and by The Gastroenterology Unit, Broadgreen
Hospital, Liverpool and were well characterised with respect to clinical,
pathological and pedigree information. Sterile blood samples were
collected from these families and lymphocytes separated out and frozen.
These were subsequently transformed by Epstein-Barr Virus (EBV) to produce
a permanent source of DNA.sup.9. The sediment of these mononucleic cell
separations was also used to provide an immediate source of DNA (legend
FIG. 1.). Five polymorphic DNA probes assigned to chromosome 5 and mostly
not regionally localised, were tested on up to 124 members from 13
different families. One of these, C11p11 (cloned by T. Robbins and kindly
provided by R. Williamson) from flow sorted human chromosome sample by the
method of T. C. Gilliam et al Human Genetics, 68, 154-158 (1984) showed
evidence for close linkage to FAP. This probe is polymorphic with TagI,
producing alleles at 3.9 kb and 4.4 kb with frequencies of about 0.92 and
0.08 (see FIG. 1). Six of the families, including 79 typed individuals,
were informative and these gave a combined maximum lod score of 3.26 at a
recombination fraction of 0=0. The lod scores for the linkage of FAP to
L1.4.sup.10 are also given and although not significant, give an estimate
for the recombination fraction .theta., of about 0.1 (see Table O). The
other probes (L1.7 from P. Pearson; .lambda.MS8 from A. Jeffreys.sup.11
and pHexXbaI from R. Gravel) showed no significant linkage with FAP or
with each other. There was no obvious heterogeneity between the families
with respect to the linkage of C11p11 with FAP. Assigning the most
probable genotypes in all cases and assuming close linkage, the data are
consistent with no recombinants amongst 24 meioses with no genotype
predictions being necessary in 12 out of these 24 meioses.
In order to relate the linkage data to the interstitial deletion reported
by Herrera et al..sup.8, in situ hybridisation was carried out to localise
C11p11 and L1.4 on chromosome 5. Probe C11p11 localises to 5q21-22 (FIG.
2a) which is consistent with the deletion 5q15-22 of Herrera et al.,
rather than their cytogenetically indistinguishable alternative 5q13-15.
This localisation is also consistent with a case report of a large bowel
adenocarcinoma which has a 5q12!-22 deletion in addition to other
cytogenetic abnormalities.sup.12. The lack of significant linkage between
HEXB or .lambda.MS8 and FAP is consistent with their localisations on the
long arm of chromosome 5 (FIG. 2b). In the case of L1.4 this may be due to
the small number of informative families rather than its localisation.
The demonstration in Example 2 that more than 20% of sporadic colorectal
carcinomas become homo- or hemi-zygous for chromosome 5 alleles, the
appreciable linkage with C11p11 and the coincidental localisation of the
probe with the two deletions.sup.8,12 provides overwhelming evidence for
assignment of FAP to chromosome 5 probably to 5q21-22.
The marker loss data, together with the localisation of FAP, extend
Knudson's ideas to FAP and, at least, a major subset of colorectal
carcinomas. The data thus suggest that becoming recessive for a gene
around 5q21-22 is a key step in the progression of many colorectal
carcinomas, both sporadic and familial.
TABLE 0
______________________________________
Lod scores from two point analyses for linkage between
C11p11, L1.4 and FAP
Recombination fraction (.theta.)
Marker loci
0.0 0.10 0.20 0.30 0.40
______________________________________
C11p11-FAP 3.26 2.56 1.88 1.15 0.44
L1.4-FAP -2.50 0.78 0.66 0.37 0.12
______________________________________
The fact that polyps from FAP patients do not yet show the recessive
change, as shown in the accompanying paper.sup.13, is consistent with data
using G6PD markers which suggested that polyps were not clonal.sup.14.
One possibility of how heterozygosity for a deficiency can give rise to
localised growth abnormalities such as the polyps is through a threshold
effect involving, for example, negative control over the production of
growth factors. In this model, the normal homozygote produces enough of
the FAP gene product so that random fluctuations of its level do not, or
at least only vary rarely, go below a threshold that permits localised
excessive growth. The deficient heterozygote however, may allow random
fluctuations in the production of FAP gene product relatively frequently
enough for it to go below this threshold. In those localised areas of the
colon where this happens, excessive epithelial growth may be initiated
giving rise to polyps. Once initiated, this growth may persist by, for
example, some form of feedback control for production of a growth factor.
This is at least one way to explain how a gene dosage effect can give rise
to discontinuous growths i.e. the polyps which look as though they should
be clonal. Presumably the polyps, once they have arisen, provide the
opportunity for the second, namely recessive, change to take place,
followed no doubt by other changes which lead to the overt carcinoma.
The similarity in frequency of FAP throughout the world, together with the
relatively high proportion of cases (up to 4%) which may be sporadic,
namely new mutations.sup.1, suggests that most FAP families may be
different mutations. At the detailed genetic level the disease may
therefore be quite hetergeneous. The incidence of FAP is estimated to be
at least 1/10,000 and perhaps up to 1/5,000 which suggests that the
mutation rate to FAP could be as high as 1/25,000. Perhaps there are
sequences around the FAP gene which predispose to relatively high mutation
rates by, for example, encouraging small deletions following unequal
intrachromosomal double crossovers or gene conversion-like events.
Now that the FAP gene has been localised, long-range DNA analysis using
pulsed field gel electrophoresis and chromosome jumping techniques.sup.15,
together with searches for appropriate epithelial specific messages coded
for in this genetic region, should lead to the identification of the FAP
gene and its function. This will not only provide a basis for the
presymptomatic or prenatal diagnosis of FAP, but also lead to approached
to counter-acting the FAP defect and may also provide new clues to
specific treatments for at least some forms of sporadic colon carcinoma.
REFERENCES.DESCRIPTION AND EXAMPLE 1
1. Bulov, S. Danish Medical Bulletin. 34(1), 1-15 (1987).
2. Veale, A. M. O. Intestinal Polyposis, Cambridge. Cambridge University
Press (1965).
3. Bussey, H. J. R. Familial polyposis coli. Baltimore. John Hopkins
University Press, 1975.
4. Gardinar, E. Am. J. Hum. Genet. 3, 167-76 (1951).
5. Muto, T. et al 36(6), 2251-70 (1975).
6. Morson, B. C. Clinical Radiology 35, 425-31 (1984).
7. Knudson, A. G. Jnr. Proc. Natl. Acad. Sci. U.S.A. 68, 820-823 (1971).
8. Herrera, L. et al. Am. J. Med. Genet. 25, 473-476 (1986).
9. Miller & Lipman. Proc. Natl. Acad. Sci. U.S.A. 70, 190-194 (1973).
10. 8th Int. Workshop on Human Gene Mapping. Cyto. Cell Genet. 40(1-4),
1-824 (1985).
11. Wong Z. et al. Ann. Hum. Genet. 51, 269-288 (1987).
12. Ferti-Passantonopoulpu et al. Cancer Genetc. Cytogenet 21, 361-4
(1986).
13. Solomon. F. et al, Nature 328, 618-619 (1987).
14. Hsu, S. H. et al. Science 221, 951-953 (1983).
15. Poustka, A. et al Nature 325, 353-355 (1987). Membrane transver and
detection methods. Amersham (1985).
16. Stoker, N. Analytical Biochemistry 137, 266-267 (1984).
17. Smith, C. A. B. Am. J. Hum. Genet. 11(4), 289-304 (1959).
18. Ott, J. Am. J. Hum. Genet. 20, 588-597 (1974).
Example 2
The dominantly inherited disorder, Familial Adenomatous Polyposis (FAP or
Familial Polyposis Coli), which gives rise to multiple adenomatous polyps
in the colon that have a relatively high probability of progressing to a
malignant adenocarcinoma.sup.11 provides a basis for studying the role of
recessive genes in the far more common colorectal carcinomas using this
approach. Following a clue as to the location of the FAP gene given by a
case report of an individual with an interstitial deletion of chromosome
5q, who had FAP and multiple developmental abnormalities.sup.12, we have
examined sporadic colorectal adinocarcinomas for loss of alleles on
chromosome 5. Using a highly polymorphic "minisatellite" probe.sup.13
which maps to chromosome 5q we have shown that at least 20% of this highly
heterogeneous set of tumours loose one of the alleles present in matched
normal tissue. This parallels the assignment of the FAP gene to chromosome
5 (see accompanying paper.sup.14) and suggests that becoming recessive for
this gene may be a critical step in the progression of a relatively high
proportion of colorectal cancers.
Tumour material and adjacent normal mucosa samples were collected from
about 45 cases of colorectal carcinomas, mostly from St. Mark's Hospital,
London. DNA preparations from these matched normal/tumour pairs were
analysed from RFLP difference using a variety of probes that had been
assigned to particular chromosomes. Following the clue that the FAP gene
might be on chromosome 5, attention was concentrated on probe
.lambda.MS8.sup.13 which is a locus-specific mini-satellite probe that
maps to chromosome 5q34-qter(N. J. Royle and A. J. Jeffrey, unpublished).
Approximately 90% of individuals (55 out of 60 in our data) are
heterozygous with this probe, showing two single band alleles with a HinfI
digests. This high level of heterozygosity makes it particularly valuable
for these types of studies. Forty four heterozygous normal/tumour pairs
were examined using HinfI digests with this probe (Table 1A). Of these,
seven tumours show clear alleles loss (nos. 9,17,18,19,35,37,38, for
examples see FIG. 3A) and three others showed obvious, but less extreme
changes in the ratio of one allele to the other when compared with normal
tissue (nos. 4,11,21, FIG. 3A). These latter three cases are likely to be
allele loss which is obscured by relatively large amounts of contaminating
normal tissue in the tumour sample. Overall, 23% of tumours showed loss or
alteration of an allele at this locus.
The same normal/tumour pairs were examined with a second probe, L1.4 which
maps approximately to 5q31 (see Example 1) .sup.14,15. The RFLP allele
frequencies detected with this probe, using ECoR1 are, on our data, 0.27
an d0.73. Seventeen informative pairs were studied and, of these, four
showed clear allele loss (nos. 5,19,28,35, FIG. 3A) while two others
showed an obvious change in the ratio of one allele to the other (nos.
9,11). Again, ambiguous results are probably due to contaminating normal
tissue in the tumour sample. The overall results with this probe, namely 6
presumed losses out of 17, were similar to those with .lambda.MS8. There
are not enough data to analyse any possible relationship between Dukes
stage or tumour type or location and allele loss.
TABLE 1a
__________________________________________________________________________
Allele losses in colorectal carcinomas
with chromosome 5 probes .lambda.MS8 and L1.4
PROBES
.lambda.MS8
Hinf I
(no. alleles L1.4
TUMOUR DUKES detected)
Eco RI
PATIENT
TYPE STAGING.sup.20
N T N T
__________________________________________________________________________
1 Ad. Ca. Rectum C1 2 2 12
12
2 Ad. Ca. Sigmoid Colon
B 2 2 12
3 Ad. Ca. Rectum C1 2 2
4 Ad. Ca. Rectum C1 2 (1)
--
--
5 Ad. Ca. Rectum C1 -- -- 12
2
6 Ad. Ca. Rectum B 2 2 12
12
7 Ad. Ca. Rectum C2 2 2
8 Ad. Ca. Rectum B 2 2 --
--
9 Ad. Ca. Sigmoid Colon
B 2 1 12
(2)
10 Ad. Ca. Rectum A 2 2 --
--
11 Ad. Ca. Rectum A 2 (1)
12
(1)
12 Ad. Ca. Sigmoid Colon
C1 2 2 --
--
13 Ad. Ca. Rectum C1 2 2 12
14 Ad. Ca. Rectum B 2 2 --
--
15 Ad. Ca. Asc. Colon
B 2 2 12
12
16 Ad. Ca. Rectum C1 2 2 --
--
17 Mucinous Ad. Ca. Rectum
C1 2 1 --
--
18 Ad. Ca. Rectum B 2 1 --
--
19 Ad. Ca. Rectum B 2 1 12
2
20 Ad. Ca. Rectum C1 2 2 --
--
21 Ad. Ca. Sigmoid Colon
B 2 (1)
12
12
22 Mucinous Ad. Ca. Asc. Colon
B 2 2 12
12
23 Ad. Ca. Rectum B 2 2 --
--
24 Ad. Ca. Rectum B 2 2 --
--
25 Ad. Ca. Sigmoid Colon
B 2 2 --
--
26 Ad. Ca. Rectum C1 2 2 --
--
27 Ad. Ca. Rectum C1 2 2 --
--
28 Ad. Ca. Rectum A 2 2 12
2
29 Ad. Ca. Asc. Colon
C1 2 2 --
--
30 Ad. Ca. Rectum C1 2 2 --
--
31 Mucinous Ad. Ca. Asc. Colon
B 2 2 --
--
32 Ad. Ca. Rectum B 2 2 --
--
33 Ad. Ca. Rectum C1 2 2 --
--
34 Ad. Ca. Sigmoid Colon
B 2 2 --
--
35 Ad. Ca. Sigmoid Colon
B 2 1 12
2
36 Ad. Ca. Rectum B 2 2 --
--
37 Ad. Ca. Sigmoid Colon
B 2 1 --
--
38 Ad. Ca. Sigmoid Colon
B 2 1 --
--
39 Ad. Ca. Rectum C1 2 2 12
12
40 Ad. Ca. Sigmoid Colon
C1 2 2 12
12
41 Ad. Ca. Rectum B 2 2 --
--
42 Ad. Ca. Desc. Colon
B 2 2 12
12
43 Ad. Ca. Desc. Colon
C 2 2 12
12
44 Ad. Ca. Rectum B 12
12
45 Ad. Ca. Asc. Colon
B 2 2 12
12
__________________________________________________________________________
TABLE 1b
__________________________________________________________________________
Combined .lambda. MS8 and L1.4 allele losses
in cases where both probes are informative
.lambda.MS8
L1.4
.lambda.MS8
L1.4
.lambda.MS8
L1.4
.lambda.MS8
L1.4
no loss
no loss
no loss
loss
loss
no loss
loss loss
Total
__________________________________________________________________________
9 1 1 4 15
__________________________________________________________________________
TABLE 1
Ad.Ca. - Adenocarcinoma; Asc. - ascending Desc. - descending. Tumour
material and adjacent normal mucosa from colorectal cancer patients,
obtained during surgery, was frozen immediately in liquid nitrogen. DNA
was analysed by standard methods, see legend to FIG. 1. .lambda.MS8
detects alleles of different sizes in almost every individual so that each
sample is indicated as having 2 or 1 alleles rather than particular allele
sizes. The L1.4 alleles are 0.7 kb (allele 1) and 0.6 kb (allele 2). "-"
indicates non-informative samples, due to lack of heterozygosity. A blank
space indicates no information. Those tumour samples with less certain
allele loss are shown with the most prominent remaining allele in
parenthesis.
The results for cases in which both markers .lambda.MS8 and L1.4 were
informative are summarised in Table 1B. A total of 6/15, or 40%, showed
changes for one or other marker, although with such small numbers, this is
not significantly higher than the 10/44 for .lambda.MS8 alone. Four out of
six were changed for both markers, suggesting a high frequency of overall
chromosomal loss. A more localised deletion or somatic recombination,
would explain case number 28 with loss at L1.4 but not .lambda.MS8. One
case, number 21, with loss at .lambda.MS8 but not L1.4 is inconsistent
with these interpretations given the presumed map order
centromere-FAP-L1.4-.lambda.MS8 (see accompanying paper). This could, once
again, be the result of misclassification for one of the markers due to
contaminating normal tissue in the tumour material.
In order to obtain a more objective quantitative assessment of the allele
ratios in the material given in Table 1, the autoradiographs for
.lambda.MS8 were scanned densitometrically. From the scans, the ratios of
the amounts of the two alleles in tumour material over those in the
matched tissues could be estimated. For true allele loss, this derived
ratio should be 0, but experimental error and normal tissue contaminating
the tumour will lead to a distribution which should be bimodal.
Preliminary results support this prediction and indicate nearly 40% (15
out of 39 cases scanned) have tumour/normal ratios in the lower part of
this distribution, FIG. 4. This suggests that a more careful assessment of
allele loss might lead to a higher estimate of the proportion of tumours
which have lost a chromosome 5 allele. Those tumours showing allele loss
which was easily detected by eye on the autoradiographs are indicated by
circles in FIG. 4.
To determine whether the loss of alleles on chromosome 5 is specific and
not simply a reflection of random loss, polymorphic markers on 6 other
chromosomes were tested, namely 7,8,11,12,13,17. These were chosen because
changes in chromosomes 7,8,12 and 17 have been reported in colorectal
tumours.sup.16-19. These tumours are difficult to karyotype and in general
show gross rearrangements and aneuploidy with no very specific chromosomal
changes. Chromosomes 13 and 11 were tested because of their roles of
retinoblastoma and Wilm's tumours respectively. The results for these
other markers are shown in table 2. In only one case was heterozygosity
not maintained, and this involved one tumour out of 21 which showed the
loss of an allele on chromosome 12. In addition, ten informative
normal/tumour pairs associated with the inherited cancer syndrome
MEN.sub.2 showed no loss of alleles on chromosome 5 using probe
.lambda.MS8 (C. Mathew, pers comm). Thus, the loss of alleles appears to e
chromosome-specific and possibly specific to this tumour.
Ten established cell lines derived from sporadic colonic adenocarcinomas
and from a polyp from an FAP case also were tested with probe .lambda.MS8.
For the colorectal lines, matching normal tissue was not available, but
since the predicted frequency of homozygotes is only 1 in 10, any increase
beyond this could be significant. The data in FIG. 3B show that 4of these
ten lines are clearly homozygous nos. 5,7,9 and 10) and a fifth most
probably so, (no. 6) strongly suggesting loss of a chromosome 5 as a
significant genetic change in a remarkably high proportion of these
tumours. The data on allele loss from cell lines are much clearer than for
the fresh tumour material, as might be expected, since for the lines there
is no problem of contamination by normal tissue. The Chi square
TABLE 2
__________________________________________________________________________
Heterozygosity in colorectal carcinomas with
chromosome-specific probes
Probe No. of patients
No. of heterozygotes
Chromosome
(Enz. detecting RFLP)
tested normal
tumour
Ref.
__________________________________________________________________________
7 D7S8 (MspI) 17 9 9 21
p.lambda.g3 (HinfI)
9 9 9 13
8 D8S8 (Taq I)
33 14 14 22
11 D11S16 (MspI)
39 19 19 23
12 pPstI (HindIII)
31 17 16 24
D12S2 (EcoRI)
27 4 4 25
13 D13S1 (MspI)
26 13 13 26
D13S2 (MspI)
44 24 24 26
D13S4 (MspI)
37 22 22 26
17 ERBA1 (PvuII)
20 1 1 27
D17S1 (MspI)
41 15 15 28
__________________________________________________________________________
Table 2
Tumour and normal DNA from colorectal cancer patients, hybridized with
probes on chromosomes 7, 8, 11, 12, 13 and 17. Methods are as described in
FIG. 1. DNA was digested with the enzymes indicated in parenthesis next to
the probe name. Patients showing loss of chromosome 5 alleles (Table 1)
were informative, but showed no loss for other probes as follows: patient
4--D7S8, D13S2; patient 5--D13S1, D17S1; patient 9--D11S16, D12S2, D17S1;
patient 11--D13S4; patient 17--p.lambda.g3; patient 18--D13S2, D17S1;
patient 19--D13S2, D13S4; patient 21--D12S2, D12S2; patient 28--D11S16;
D13S2, D13S4, D17S1. (X.sup.2) is 9 for the difference between 5/10 and
the control .lambda.MS8 homozygote frequency of 5/60 and this is highly
significant with pz0.35.
One of the cell lines showing .lambda.MS8 allele loss, PC/JW.sup.19 was
derived from a carcinoma from an FAP patient, which is of course
consistent with the mapping of the FAP gene to chromosome 5. However, now
that FAP patients are treated removal of the colon at a relatively early
age to prevent cancers developing, it is not easy to obtain colorectal
tumour material from FAP patients. We have, therefore, not yet been able
to extend our observations to a larger number of familial cases of
colorectal tumours. The cell line PC/AA.sup.19, derived from a polyp, did
not show loss of an .lambda.MS8 allele. This is consistent with tests,
using .lambda.MS8 of premalignant polyps from 11 FAP patients, which
showed no loss of heterozygosity in any case. The recessive change on
chromosome 5 is, thus, secondary to the initial development of a polyp
(see Example 1 for further discussion).
The overall data clearly show that a relatively high proportion of sporadic
colorectal carcinomas loose all or part of one chromosome 5, presumably
because loss of the FAP gene function favours tumour progression. The fact
that .lambda.MS8 which is near the tip of 5q, demonstrated this loss,
together with the comparatively common concominant loss of an allele of
the L1.4 locus, suggests that whole chromosome loss by non-disjunction,
presumably following an initial mutation on one of the two chromosomes 5,
is the commonest mechanism for becoming recessive, as has been found, for
example, for retinoblastoma.sup.2. It would be expected, that this loss
would be accompanied by reduplication since monosomy is likely to be
disadvantageous for the growth of any cell, and non-disjunction is a
relatively common event. Preliminary karyotyping of several cell lines
supports this (data not shown). Even if technical developments increase
the proportion of tumours with chromosome 5 allele loss to around 40%,
there will still be a sizeable fraction of tumours in which no change on
chromosome 5 is readily detectable. This could in part, of course, be due
to relatively localised genetic mechanisms for producing the secondary
change, such as point mutations, or small delections, or somatic crossing
over. There is however, the further important likelihood that colorectal
tumours are aetiologically heterogeneous with respect to the role of the
FAP gene on chromosome 5. Those in which this activity is lost may
represent a particular sub-set of colorectal tumours and this may have
important implications for diagnosis and treatment. The combined studies
of this and the accompanying paper on the localisation of the FAP gene and
its probable role in a relatively high proportion of colorectal
carcinomas, uncovers for the first time an important recessive genetic
effect in one of the commonest of all cancers.
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